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• W3092862007 abstract "Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy. Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy. Gliomas are tumors that occur in the brain and spinal cord. Glioma, which begins in the glial cells that surround neurons and help them function, is one of the most common types of malignant primary brain tumors. Treatment for glioma depends on the cell type, size, grade of malignancy, and location of the tumor. Treatment is usually a combination of surgery, radiation therapy, chemotherapy, and immunotherapy. Targeted drug therapy is preferred over conventional cytotoxic chemotherapy, as the targeted drug treatments focus on specific abnormalities present within cancer cells (1Le Rhun E. Preusser M. Roth P. Reardon D.A. van den Bent M. Wen P. Reifenberger G. Weller M. Molecular targeted therapy of glioblastoma.Cancer Treat. Rev. 2019; 80 (31541850)10189610.1016/j.ctrv.2019.101896Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2Miller J.J. Wen P.Y. Emerging targeted therapies for glioma.Expert Opin. Emerg. Drugs. 2016; 21 (27809598): 441-45210.1080/14728214.2016.1257609Crossref PubMed Scopus (40) Google Scholar). By blocking these abnormalities, targeted drug treatments can cause cancer cells to die without affecting noncancer cells (3Mooney J. Bernstock J.D. Ilyas A. Ibrahim A. Yamashita D. Markert J.M. Nakano I. Current approaches and challenges in the molecular therapeutic targeting of glioblastoma.World Neurosurg. 2019; 129 (31152883): 90-10010.1016/j.wneu.2019.05.205Abstract Full Text PDF PubMed Scopus (19) Google Scholar). However, for the targeted drug therapy to be successfully developed and used in the clinic, one needs to better understand glioma cell behaviors (such as glioma cell migration/invasion, growth/proliferation, death/survival, and drug resistance) and related signaling pathways. The signaling component “p21-activated kinase-1” (PAK1) is a serine/threonine kinase regulated by small GTP-binding proteins Cdc42 and Rac (4Zhao Z.S. Manser E. PAK family kinases: physiological roles and regulation.Cell Logist. 2012; 2 (23162738): 59-6810.4161/cl.21912Crossref PubMed Google Scholar, 5Teramoto H. Crespo P. Coso O.A. Igishi T. Xu N. Gutkind J.S. The small GTP-binding protein Rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells: evidence for a Pak-independent signaling pathway.J. Biol. Chem. 1996; 271 (8824197): 25731-2573410.1074/jbc.271.42.25731Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Ong C.C. Jubb A.M. Zhou W. Haverty P.M. Harris A.L. Belvin M. Friedman L.S. Koeppen H. Hoeflich K.P. p21-activated kinase 1: PAK'ed with potential.Oncotarget. 2011; 2 (21653999): 491-49610.18632/oncotarget.271Crossref PubMed Google Scholar, 7Taglieri D.M. Ushio-Fukai M. Monasky M.M. P21-activated kinase in inflammatory and cardiovascular disease.Cell. Signal. 2014; 26 (24794532): 2060-206910.1016/j.cellsig.2014.04.020Crossref PubMed Scopus (17) Google Scholar). This kinase affects a wide variety of cellular processes, such as cell motility, invasion, metastasis, growth, cell cycle progression, and angiogenesis. It has been previously implicated in a wide range of biological processes and the development of multiple types of cancer (8van Zijl F. Krupitza G. Mikulits W. Initial steps of metastasis: cell invasion and endothelial transmigration.Mutat. Res. 2011; 728 (21605699): 23-3410.1016/j.mrrev.2011.05.002Crossref PubMed Scopus (421) Google Scholar, 9Gupta S.C. Kim J.H. Prasad S. Aggarwal B.B. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals.Cancer Metastasis Rev. 2010; 29 (20737283): 405-43410.1007/s10555-010-9235-2Crossref PubMed Scopus (575) Google Scholar, 10Kichina J.V. Goc A. Al-Husein B. Somanath P.R. Kandel E.S. PAK1 as a therapeutic target.Expert Opin. Ther. Targets. 2010; 14 (20507214): 703-72510.1517/14728222.2010.492779Crossref PubMed Scopus (75) Google Scholar, 11Bright M.D. Garner A.P. Ridley A.J. PAK1 and PAK2 have different roles in HGF-induced morphological responses.Cell. Signal. 2009; 21 (19628037): 1738-174710.1016/j.cellsig.2009.07.005Crossref PubMed Scopus (33) Google Scholar, 12Coniglio S.J. Zavarella S. Symons M.H. Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms.Mol. Cell Biol. 2008; 28 (18411304): 4162-417210.1128/MCB.01532-07Crossref PubMed Scopus (95) Google Scholar, 13Rane C.K. Minden A. P21 activated kinase signaling in cancer.Semin. Cancer Biol. 2018; 54 (29330094): 40-4910.1016/j.semcancer.2018.01.006Crossref PubMed Scopus (75) Google Scholar, 14Semenova G. Chernoff J. Targeting PAK1.Biochem. Soc. Trans. 2017; 45 (28202661): 79-8810.1042/BST20160134Crossref PubMed Scopus (26) Google Scholar). More specifically, PAK1 has been previously implicated in cell death/survival, cell migration, and glioma (15Parvathy M. Sreeja S. Kumar R. Pillai M.R. Potential role of p21 activated kinase 1 (PAK1) in the invasion and motility of oral cancer cells.BMC Cancer. 2016; 16 (27229476): 29310.1186/s12885-016-2263-8Crossref PubMed Scopus (13) Google Scholar, 16Wang G. Zhang Q. Song Y. Wang X. Guo Q. Zhang J. Li J. Han Y. Miao Z. Li F. PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion.Cell Death Dis. 2015; 6 (25766321)e168210.1038/cddis.2015.50Crossref PubMed Google Scholar, 17Ong C.C. Jubb A.M. Haverty P.M. Zhou W. Tran V. Truong T. Turley H. O'Brien T. Vucic D. Harris A.L. Belvin M. Friedman L.S. Blackwood E.M. Koeppen H. Hoeflich K.P. Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells.Proc. Natl. Acad. Sci. U. S. A. 2011; 108 (21482786): 7177-718210.1073/pnas.1103350108Crossref PubMed Scopus (156) Google Scholar, 18Yuan Z.Q. Kim D. Kaneko S. Sussman M. Bokoch G.M. Kruh G.D. Nicosia S.V. Testa J.R. Cheng J.Q. ArgBP2γ interacts with Akt and p21-activated kinase-1 and promotes cell survival.J. Biol. Chem. 2005; 280 (15784622): 21483-2149010.1074/jbc.M500097200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Aoki H. Yokoyama T. Fujiwara K. Tari A.M. Sawaya R. Suki D. Hess K.R. Aldape K.D. Kondo S. Kumar R. Kondo Y. Phosphorylated Pak1 level in the cytoplasm correlates with shorter survival time in patients with glioblastoma.Clin. Cancer Res. 2007; 13 (18006760): 6603-660910.1158/1078-0432.CCR-07-0145Crossref PubMed Scopus (46) Google Scholar). The expression level of PAK1 has been associated with the invasiveness of glioblastoma and survival time in patients with glioblastoma. Our own previous RNAi selection study indicated that the PAK kinase family regulates cell migration (20Seo M. Lee S. Kim J.H. Lee W.H. Hu G. Elledge S.J. Suk K. RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways.Nat. Commun. 2014; 5 (25347953)521710.1038/ncomms6217Crossref PubMed Scopus (18) Google Scholar). In this study, to better understand PAK1 kinase signaling pathways and to uncover new drug targets related to these pathways, we sought to exploit a model eukaryote which is amenable to rapid genome-scale experimentation to identify related functions. More specifically, we performed a genetic interaction screen for yeast deletions that can relieve the toxic effects of PAK1 expression in yeast, thereby identifying orthologous human genes as candidate genetic interaction partners. This is based on our previous study, in which overexpression of human PAK1 was toxic in yeast (21Kim J.H. Seo Y. Jo M. Jeon H. Lee W.H. Yachie N. Zhong Q. Vidal M. Roth F.P. Suk K. Yeast-based genetic interaction analysis of human kinome.Cells. 2020; 9 (32392905)115610.3390/cells9051156Crossref Scopus (2) Google Scholar). Subsequent mammalian cell- and tissue-based validation experiments of these candidates identified multiple determinants of glioma cell migration, drug resistance, and proliferation, helping to elucidate the molecular mechanisms of PAK1-mediated carcinogenesis and other cellular behaviors. Functional analysis of kinase signaling pathways can be achieved by mapping genetic interaction networks for kinase genes (22Youn J.Y. Friesen H. Nguyen Ba A.N. Liang W. Messier V. Cox M.J. Moses A.M. Andrews B. Functional analysis of kinases and transcription factors in Saccharomyces cerevisiae using an integrated overexpression library.G3 (Bethesda). 2017; 7 (28122947): 911-92110.1534/g3.116.038471Crossref PubMed Scopus (9) Google Scholar, 23Lee K.T. So Y.S. Yang D.H. Jung K.W. Choi J. Lee D.G. Kwon H. Jang J. Wang L.L. Cha S. Meyers G.L. Jeong E. Jin J.H. Lee Y. Hong J. et al.Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans.Nat. Commun. 2016; 7 (27677328)1276610.1038/ncomms12766Crossref PubMed Scopus (64) Google Scholar, 24Sharifpoor S. van Dyk D. Costanzo M. Baryshnikova A. Friesen H. Douglas A.C. Youn J.Y. VanderSluis B. Myers C.L. Papp B. Boone C. Andrews B.J. Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs.Genome Res. 2012; 22 (22282571): 791-80110.1101/gr.129213.111Crossref PubMed Scopus (49) Google Scholar). Here we sought genes for which perturbation modifies the activity of PAK1 kinase. Because Saccharomyces cerevisiae has been a faithful model for many conserved aspects of eukaryotic cell biology (25Botstein D. Fink G.R. Yeast: an experimental organism for 21st century biology.Genetics. 2011; 189 (22084421): 695-70410.1534/genetics.111.130765Crossref PubMed Scopus (305) Google Scholar), and because genetic interaction screens can be efficiently carried out in yeast, we used a pool of yeast deletion mutants that may modify the PAK1 toxicity phenotype. To identify PAK1 genetic interactions, we previously performed a genome-wide pooled screen to identify genetic interactions on the basis of toxicity modification (21Kim J.H. Seo Y. Jo M. Jeon H. Lee W.H. Yachie N. Zhong Q. Vidal M. Roth F.P. Suk K. Yeast-based genetic interaction analysis of human kinome.Cells. 2020; 9 (32392905)115610.3390/cells9051156Crossref Scopus (2) Google Scholar). In that screen, the PAK1 gene was first introduced into a pool of 4,653 homozygous diploid yeast deletion mutants such that each mutant harbors unique barcode sequences flanking the deletion locus (26Giaever G. Shoemaker D.D. Jones T.W. Liang H. Winzeler E.A. Astromoff A. Davis R.W. Genomic profiling of drug sensitivities via induced haploinsufficiency.Nat. Genet. 1999; 21 (10080179): 278-28310.1038/6791Crossref PubMed Scopus (448) Google Scholar). Second, PAK1 gene expression was induced in yeast cells grown on galactose media. Third, yeast barcodes were amplified from deletion pool cultures. Finally, yeast barcode abundances were quantified using next-generation sequencing (Bar-Seq) to identify fitness values of PAK1 genetic interactions (27Smith A.M. Durbic T. Kittanakom S. Giaever G. Nislow C. Barcode sequencing for understanding drug-gene interactions.Methods Mol. Biol. 2012; 910 (22821592): 55-6910.1007/978-1-61779-965-5_4Crossref PubMed Scopus (8) Google Scholar). The relative abundance of each yeast barcode is a proxy for differential growth of the corresponding deletion strain, which allowed us to detect modulation of PAK1 toxicity in the absence of a specific yeast gene (28Smith A.M. Heisler L.E. Mellor J. Kaper F. Thompson M.J. Chee M. Roth F.P. Giaever G. Nislow C. Quantitative phenotyping via deep barcode sequencing.Genome Res. 2009; 19 (19622793): 1836-184210.1101/gr.093955.109Crossref PubMed Scopus (200) Google Scholar, 29Smith A.M. Heisler L.E. St Onge R.P. Farias-Hesson E. Wallace I.M. Bodeau J. Harris A.N. Perry K.M. Giaever G. Pourmand N. Nislow C. Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.Nucleic Acids Res. 2010; 38 (20460461): e14210.1093/nar/gkq368Crossref PubMed Scopus (213) Google Scholar). For each of 4,653 yeast deletions, we identified the relative abundance of each deletion strain after selection in the presence of PAK1. Genetic interactions were identified on the basis of Z-scores: Z-scores >1.96 were identified as toxicity suppressors. Z-score of 1.96 corresponds to confidence level 95% and p value <0.05, which is commonly used for statistical significance (21Kim J.H. Seo Y. Jo M. Jeon H. Lee W.H. Yachie N. Zhong Q. Vidal M. Roth F.P. Suk K. Yeast-based genetic interaction analysis of human kinome.Cells. 2020; 9 (32392905)115610.3390/cells9051156Crossref Scopus (2) Google Scholar, 30Jo M. Chung A.Y. Yachie N. Seo M. Jeon H. Nam Y. Seo Y. Kim E. Zhong Q. Vidal M. Park H.C. Roth F.P. Suk K. Yeast genetic interaction screen of human genes associated with amyotrophic lateral sclerosis: identification of MAP2K5 kinase as a potential drug target.Genome Res. 2017; 27 (28596290): 1487-150010.1101/gr.211649.116Crossref PubMed Scopus (6) Google Scholar). Among the 402 yeast suppressors of PAK1 toxicity, 131 yeast genes had human orthologs in our previous study (21Kim J.H. Seo Y. Jo M. Jeon H. Lee W.H. Yachie N. Zhong Q. Vidal M. Roth F.P. Suk K. Yeast-based genetic interaction analysis of human kinome.Cells. 2020; 9 (32392905)115610.3390/cells9051156Crossref Scopus (2) Google Scholar). Human orthologs of the yeast toxicity suppressors were identified using the Karolinska Institute's InParanoid Database (31O'Brien K.P. Remm M. Sonnhammer E.L. Inparanoid: a comprehensive database of eukaryotic orthologs.Nucleic Acids Res. 2005; 33 (15608241): D476-D48010.1093/nar/gki107Crossref PubMed Scopus (541) Google Scholar). Of these 131 genes, 19 genes (15%) that had human orthologs and were expressed in glioma cells were selected for further evaluation in the current study (Table 1 and Fig. S1).Table 1List of PAK1 genetic interactions expressed in glioma cellsNo.Yeast gene namesUniProt IDDescriptionHuman orthologs1YMR246WFAA4P47912Long-chain fatty acid–CoA ligase 4, EC 6.2.1.3 (fatty acid activator 4) (long-chain acyl-CoA synthetase 4)ACSL42YHR028CDAP2P18962Dipeptidyl aminopeptidase B, DPAP B, EC 3.4.14.– (YSCV)DPP43YCL011CGBP2P25555Single-strand telomeric DNA–binding protein GBP2, G-strand–binding protein 2 (RAP1 localization factor 6)ELAVL14YBL003CHTA2P04912Histone H2A.2H2AFX5YBR010WHHT1P61830Histone H3H3F3A6YAL005CSSA1P10591Heat shock protein SSA1 (heat shock protein YG100)HSPA87YEL009CGCN4P03069General control protein GCN4 (amino acid biosynthesis regulatory protein)JUNB8YBL063WKIP1P28742Kinesin-like protein KIP1 (chromosome instability protein 9)KIF119YER007C-ATMA20P89886Translation machinery–associated protein 20MCTS110YBL091CMAP2P38174Methionine aminopeptidase 2, MAP 2, MetAP 2, EC 3.4.11.18 (peptidase M)METAP211YJR066WTOR1P35169Serine/threonine-protein kinase TOR1, EC 2.7.11.1 (dominant rapamycin resistance protein 1) (phosphatidylinositol kinase homolog TOR1) (target of rapamycin kinase 1)mTOR12YPL006WNCR1Q12200NPC intracellular cholesterol transporter 1–related protein 1 (Niemann–Pick type C–related protein 1)NPC113YBL024WNCL1P38205Multisite-specific tRNA:(cytosine-C(5))-methyltransferase, EC 2.1.1.202 (multisite-specific tRNA:m5C-methyltransferase) (tRNA (cytosine-5-)-methyltransferase NCL1) (tRNA methyltransferase 4)NSUN214YOR347CPYK2P52489Pyruvate kinase 2, PK 2, EC 2.7.1.40PKM1, PKM215YJR100CAIM25P47140Altered inheritance rate of mitochondria protein 25PLSCR116YJL134WLCB3P47013Dihydrosphingosine-1-phosphate phosphatase LCB3, EC 3.1.3.- (long-chain base protein 3) (sphingolipid resistance protein 2)SGPP117YHR114WBZZ1P38822Protein BZZ1 (LAS17-binding protein 7)TRIP1018YCR008WSAT4P25333Serine/threonine-protein kinase HAL4/SAT4, EC 2.7.11.1 (halotolerance protein 4)TTK19YER177WBMH1P29311Protein BMH1YWHAE Open table in a new tab For functional evaluation of the PAK1 genetic interactions, siRNA-mediated knockdown in glioma cells was performed for these 20 orthologs of yeast interaction partners (note that, for yeast PYK2, we tested two orthologs, PKM1 and PKM2). Five siRNAs were designed for each PAK1 interaction partner (see Table S1 for siRNA sequences and Fig. S1 for knockdown efficiency). In cases where all five siRNAs were ineffective in knocking down the expression of the corresponding gene, an additional siRNA was designed and synthesized. We tested whether the expression knockdown of candidate genes could modify PAK1-induced cell migration and death/survival (see Fig. S2 for knockdown efficiency of siRNAs in PAK1 transfectants). Cell migration was first assessed using GL26 mouse glioma cells stably overexpressing PAK1 (Fig. S3). Because cells expressing either WT or a constitutively active mutant PAK1 cDNA showed similar PAK1 phosphorylation levels (Fig. S4) and enhanced cell migration phenotypes (Fig. S5), further experiments were performed only with WT PAK1-expressing GL26 cells (32Kim H. Oh J.Y. Choi S.L. Nam Y.J. Jo A. Kwon A. Shin E.Y. Kim E.G. Kim H.K. Down-regulation of p21-activated serine/threonine kinase 1 is involved in loss of mesencephalic dopamine neurons.Mol. Brain. 2016; 9 (27121078): 4510.1186/s13041-016-0230-6Crossref PubMed Scopus (11) Google Scholar). We could not determine exactly whether exogenous PAK1 expression affected endogenous PAK1 activity. Overexpression of WT kinases often induces or enhances their own activity through autophosphorylation, etc. as a result of a potential “artifact” of overexpression (33Lun X.K. Szklarczyk D. Gabor A. Dobberstein N. Zanotelli V.R.T. Saez-Rodriguez J. von Mering C. Bodenmiller B. Analysis of the human kinome and phosphatome by mass cytometry reveals overexpression-induced effects on cancer-related signaling.Mol. Cell. 2019; 74 (31101498): 1086-1102.e510.1016/j.molcel.2019.04.021Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 34Ishikawa E. Kosako H. Yasuda T. Ohmuraya M. Araki K. Kurosaki T. Saito T. Yamasaki S. Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1.Nat. Commun. 2016; 7 (27670070)1275610.1038/ncomms12756Crossref PubMed Scopus (21) Google Scholar, 35Camurdanoglu B.Z. Hrovat C. Dürnberger G. Madalinski M. Mechtler K. Herbst R. MuSK kinase activity is modulated by a serine phosphorylation site in the kinase loop.Sci. Rep. 2016; 6 (27905551)3827110.1038/srep38271Crossref PubMed Scopus (0) Google Scholar). Thus, under the overexpression conditions, WT and a constitutively active mutant form of PAK1 might have exhibited similar levels of activation. We used two stable cell clones, GL26-PAK1-1 and GL26-PAK1-2, that each showed PAK1 overexpression and activation as indicated by phosphorylation. Overexpression of kinases is commonly used to determine the role of kinases in cancer-related signaling (33Lun X.K. Szklarczyk D. Gabor A. Dobberstein N. Zanotelli V.R.T. Saez-Rodriguez J. von Mering C. Bodenmiller B. Analysis of the human kinome and phosphatome by mass cytometry reveals overexpression-induced effects on cancer-related signaling.Mol. Cell. 2019; 74 (31101498): 1086-1102.e510.1016/j.molcel.2019.04.021Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Among the 5–6 siRNAs for each interaction partner, the one with the best knockdown efficiency was introduced into PAK1-transfected GL26 cells, and cell migration was assessed by a wound-healing assay. Among the 20 PAK1 interaction partners, knockdown of seven genes (DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE) attenuated PAK1-induced cell migration, indicating that these genes related to proteases, mitotic spindle, autophagy, and metabolism of glucose and sphingolipids genetically interact with PAK1 to affect cell migration (Table 2 and Fig. S6). The cell migration assays were performed under the condition of no significant cytotoxicity (Fig. S7).Table 2Evaluation of candidate PAK1 human genetic interactions with respect to cell migration using siRNAsSerial no.Gene namesiRNA no.Migration assayaGenetic interaction with p value tested by wound-healing assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; −, not significant).GL26-PAK1-1GL26-PAK1-21ACSL45−−2DPP41++3ELAVL15−−4H2AFX1−−5H3F3A1−−6HSPA81−−7JUNB2−−8KIF115++9MCTS11−−10METAP21−−11mTOR1++12NPC11−−13NSUN23−−14PKM14−−15PKM26++16PLSCR14−−17SGPP11++18TRIP105−−19TTK1++20YWHAE5++a Genetic interaction with p value tested by wound-healing assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; −, not significant). Open table in a new tab Pharmacological inhibitors were next used to further investigate the yeast-orthologous human genetic modifiers of PAK1 identified in the large-scale screen. Among the 20 PAK1 interaction partners tested by siRNA-mediated knockdown, pharmacological inhibitors were commercially available for six candidates. These six pharmacological inhibitors were used in wound-healing assays in a manner similar to that used for the siRNA-mediated knockdown experiments. Among the six pharmacological inhibitors, four compounds (monastrol, KIF11 inhibitor; MPS1-IN-1, TTK inhibitor; rapamycin, mTOR inhibitor; vildagliptin, DPP4 inhibitor) reduced PAK1-induced GL26 glioma cell migration, whereas two compounds (fumagillin, METAP2 inhibitor; P-M2tide, PKM2 inhibitor) had no significant effects (Table 3 and Fig. S8). All inhibitors were used at optimal concentrations without apparent cytotoxicity based on the IC50 as reported in previous studies (36Koch A. Maia A. Janssen A. 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Chem. 2007; 282 (17251189): 9740-974710.1074/jbc.M608883200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and as determined by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell viability assay (Figs. S9 and S10). The results of the pharmacological inhibitor experiments were consistent with those of the siRNA-mediated genetic knockdown experiments except for PKM2. PKM2 inhibitor P-M2tide did not significantly influence PAK1-induced cell migration, whereas siRNA-mediated knockdown of PKM2 gene expression had significant effects. This discrepancy may be due to the complex role of PKM2 expression and activity in regulating cell behaviors (40Wiese E.K. Hitosugi T. Tyrosine kinase signaling in cancer metabolism: PKM2 paradox in the Warburg effect.Front. Cell Dev. Biol. 2018; 6 (30087897): 7910.3389/fcell.2018.00079Crossref PubMed Scopus (30) Google Scholar, 41Lu Z. Hunter T. Metabolic kinases moonlighting as protein kinases.Trends Biochem. Sci. 2018; 43 (29463470): 301-31010.1016/j.tibs.2018.01.006Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 42Dayton T.L. Jacks T. Vander Heiden M.G. PKM2, cancer metabolism, and the road ahead.EMBO Rep. 2016; 17 (27856534): 1721-173010.15252/embr.201643300Crossref PubMed Scopus (186) Google Scholar).Table 3Evaluation of candidate PAK1 genetic interactions with respect to cell migration using pharmacological inhibitorsSerial no.Gene nameInhibitor nameMigration assayaGenetic interaction with p value tested by wound-healing assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; −, not significant).GL26-PAK1-1GL26-PAK1-21DPP4Vildagliptin++2KIF11Monastrol++3METAP2Fumagillin−−4mTORRapamycin++5PKM2P-M2tide−−6TTKMPS1-IN-1++a Genetic interaction with p value tested by wound-healing assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; −, not significant). Open table in a new tab For the functional evaluation of PAK1 genetic interactions with respect to cell death/survival phenotypes, glioma cells were used for anticancer drug toxicity testing and cell proliferation assays. PAK1-overexpressing GL26 glioma cells were transfected with siRNAs and then exposed to bis-chloroethylnitrosourea (BCNU), an anticancer drug that is similar to temozolomide, which is commonly used in the treatment of glioma patients. Glioma cells stably expressing PAK1 clearly showed increased drug resistance compared with that of parental cells. Knockdown of PAK1 interaction partner expression using siRNAs modulated glioma cell death (PAK1-induced drug resistance), as evaluated by lactate dehydrogenase (LDH) and MTT assays (Table 4 and Figs. S11 and S12), indicating genetic interactions between PAK1 and the knocked down genes (DPP4, mTOR, SGPP1, TTK, and YWHAE). A BCNU concentration of 200 μm was used for these experiments, based on dose-dependent toxicity test results (Fig. S13), and the mode of cell death was primarily apoptosis, as determined by a caspase-3 activity assay (Fig. S14). Differences between controls and PAK1-overexpressing cells were modest in some of the results. However, the difference was statistically significant in the key comparisons.Table 4Evaluation of candidate PAK1 genetic interactions with respect to drug resistance using siRNAsSerial no.Gene namesiRNA no.LDH assayaGenetic interaction with p value tested by LDH assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; ++, p < 0.01; −, not significant).MTT assaybGenetic interaction with p value tested by MTT assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; ++, p < 0.01; −, not significant).GL26-PAK1-1GL26-PAK1-2GL26-PAK1-1GL26-PAK1-21ACSL45−−−−2DPP41+++++3ELAVL15−−−−4H2AFX1−−−−5H3F3A1−−−−6HSPA81−−−−7JUNB2−−−−8KIF115−−−−9MCTS11−−−−10METAP21−−−−11mTOR1+++++12NPC11−−−−13NSUN23−−−−14PKM14−−−−15PKM26−−−−16PLSCR14−−−−17SGPP11++++++18TRIP105−−−−19TTK1++++++20YWHAE5+++++a Genetic interaction with p value tested by LDH assay in two independent clones of PAK1 stable transfectants (GL26-PAK1-1 and GL26-PAK1-2) (+, p < 0.05; ++, p < 0.01; −, not significant).b Genetic interaction with p value tested by MTT assay in two" @default.
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• W3092862007 title "Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways" @default.
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• W3092862007 doi "https://doi.org/10.1074/jbc.ra120.014831" @default.
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